Triose Phosphate Isomerase Structure & Mechanism

General Information
Triose phosphate isomerase (TIM) (PDB 1wyi and 1hti) is a crucial enzyme in the glycolytic pathway. TIM reversibly converts the aldose Glyceraldehyde-3-phosphate (GAP) to the ketose Dihydroxyacetone phosphate (DHAP). The interconversion proceeds by an enediol intermediate. Triose phosphate isomerase is not directly regulated, but the enzyme two steps before it in the glycolytic pathway, phosphofructokinase, is a heavily regulated, irreversible enzyme.

Structural Characteristics
The secondary structure consists of 14 alpha helices and 8 beta sheets per monomer, making it fall in the SCOP category of alpha and beta proteins. The tertiary structure is a alpha-beta barrel. The quaternary structure is a homodimer. The molecular weight of the enzyme is estimated at 57,400 Da.

Mechanism
The enzyme aids in catalysis by binding tightly to the enediol transition state. To convert GAP to the enediol intermediate, a proton is abstracted from C2 by a base and the carbonyl oxygen atom is protonated by an acid.  Glu 165 acts as the base and grabs the C2 proton on glyceraldehyde-3-phosphate, while His 95 is H-bonded to the carbonyl oxygen and acts as the acid by protonating carbonyl oxygen. The enediol intermediate is negatively charged, but is somewhat stabilized by the positively charged side chain of Lys 12. Mutation of Lys 12 to Arg increases Km by a factor of 22 and decreases Vmax by a factor of 180. To convert the enediol intermediate to DHAP, C1 is protonated by Glu 165, with His 95 removing a proton from C2’s OH group. As a result, the catalytic groups are back to their original states, and catalysis is complete. With GAP as a substrate, Km for the reaction is .34 mM and Vmax is 7200 units/mg protein at 25 degrees C and pH 7.5.



An interesting part of the enzyme is the flexible loop that stabilizes the enediol-like transition state. The flexible loop (residues 167-176) closes over the active site like a hinged lid when substrate is bound, thus preventing phosphate from leaving. A four-residue segment of the loop H-bonds with the phosphate group of the substrate. Without the loop, the enediol intermediate would eliminate phosphate, with the end products being inorganic phosphate and toxic methylglyoxal.

Additional Resources
For additional information, see: Carbohydrate Metabolism